1. Field of the Invention
The present invention concerns an improved process for the production of immobilized antibodies, with regiospecific conjugates of antibodies containing oligosaccharides being formed by means of oxidative modification of the carbohydrate region of the antibodies.
2. Discussion of the Prior Art
The related U.S. Application Ser. No. 07/270,280 now U.S. Pat. No. 4,948,836 describes immobilized antibodies which are bound covalently to a matrix polymer, specifically by means of a modification in the carbohydrate region of the antibodies. Binding of the antibodies is achieved by a condensation reaction between at least one aldehyde group in the oxidatively modified carbohydrate region and at least one epoxide function of a matrix polymer. The polymer is modified by means of a bifunctional reagent which consists of a spacer with at least a three link chain and an end group suitable for condensation with the oxidatively produced aldehyde function of the antibody. A group at the other end of the bifunctional reagent reacts covalently with the epoxy group of the polymer.
Although the technique described in the aforementioned U.S. patent application results in very usable products, there continues to be significant interest in improving the process. For example, shortening the process period and simplifying the process itself has a positive effect on the overall result given the notorious sensitivity of antibodies. Therefore, the present invention is best understood as an improved process, based on the teaching of the U.S. Pat. No. 4,948,836.
The present process is based on oxidative binding of antibodies containing oligosaccharide moieties which have been oxidized by means of periodate, to a polymer carrier (ADH-PC) modified by means of adipic acid dihydrazide, in a single-crucible reaction.
In the multi-stage method which is disclosed in the aforementioned patent application the antibodies are first oxidized with periodate, then excess reagent is removed by treatment with ethylene glycol followed by gel filtration, concentration, and finally coupling of the antibodies to the hydrazide-modified polymer carrier. In contrast, in the present new process, the antibodies are oxidized to the aldehyde stage and then incubated with the polymer carrier ADH-PC which has been modified with adipic acid dihydrazide, in a single work step and without isolation or purification of intermediates. This process allows for efficient coupling of the sensitive antibody to the polymer without physical losses of material, nor losses in binding activity of the antibody.
The present invention therefore concerns a process for the production of immobilized antibodies containing oligosaccharide, by condensation of the antibodies present in oxidized form, with a carrier ADH-PC modified with adipic acid dihydrazide. The antibodies are incubated in a single-crucible process in an aqueous medium which simultaneously contains periodate (in amounts which are sufficient for oxidation to the aldehyde in the area of the carbohydrate region of the antibodies) and the polymer carrier material modified by means of adipic acid dihydrazide.
In this way an oxidative, covalent fixation is obtained since the aldehyde groups, once they have occurred due to oxidation, react directly with the adipic acid dihydrazide (ADH) groups of the carrier ADH-PC.